GB2160870A

GB2160870A - Process for preparing peptides

Info

Publication number: GB2160870A
Application number: GB08516281A
Authority: GB
Inventors: Stefano Cambiaghi; Franco Dallatomasina; Pietro Giardino; Enzo Murador; Gaspare Spreafico
Original assignee: Farmitalia Carlo Erba SRL
Current assignee: Pfizer Italia SRL
Priority date: 1984-06-27
Filing date: 1985-06-27
Publication date: 1986-01-02
Also published as: GB2160870B; JPH0669389B2; JPS6121095A; GB8516281D0; FR2566800A1; BE902474A; US4668625A; IT8421622A1; IT1176332B; FR2566800B1; DE3523018A1; IT8421622A0

Description

1 GB 2 160 870 A 1

SPECIFICATION

Process for preparing peptides The invention relates to a process for the preparation of peptides and more particularly to a process for the preparation of lower alkyl esters of L-cx-aspartyl-L-phenylaianine by enzymatic hydrolysis of suitable N- acyl-derivatives The lower alkyl esters of L-a-aspartyl- L-phenylalanine include the methyl ester, which is the well known sweetening agent aspartame.

In the specific field of the preparation of aspartame, an N-acyl- L-(xaspartyl-L-phenylalanine methyl prepared from an acyl-L-aspartyl derivative and L-phenylalanine methyl ester, is usually hydrolyzed chemically, for example by a strong acid (US 4071511) or by a base such as hydroxylamine or acety1hydrazine (GB 2098220). However, the process of chemical hydrolysis has several disadvantages. Principally, it is aspecific and leads to the formation of by-products. Peptido hydrolasic reactions form aspartic acid and phenylalanine derivatives. Esterasic reactions form demethylated aspartames. Cyclization reac- tions form variously substituted diketopiperazines. The presence of all these by-products, besides lowering the production yields, makes it difficult to extract the desired compound at the desired degree o purity. Further chemical and biochemical processes are also needed to recover the important by-products of the reaction so as to optimize the process from an economic point of view.

The invention provides a process for the preparation of an ester of L-(xaspartyl-L-phenylaianine having 20 the general formula I COOR, 1 H2N-CH-uU-NH-uti-uH2-C,H, 1 UM2-CO01-1 (1) wherein R, represents an alkyl group having from 1 to 4 carbon atoms, the process comprising enzymatically hydrolysing an N-acyl derivative of the desired product, which N-acyl derivative has the general 30 formula 11 3E COOR, 1 R-CO-N H-CH-CO-N H-CM-t;'12-1-;J15 i (;H,-uuutl (11) wherein R represents a free or substituted alkyl, alkenyl or phenylalkyl group having from 1 to 11 carbon atoms and R, is as above defined. 40 Preferably R, represents a methyl group and R represents a hydrogen atom or a group of the formula 40 CH,, -(CH,)nCH3wherein n is a number of from 1 to 10, -C,H,, -CH,-CH,, -CH2-C,,H,- OH, -CH2-C,H,-NO,, C,H3(OCH3)2, -CH2-C,H,(OH) 21 -CH2-CH=CH-CH2-CH,-CH2-S-CH2-CH=CH, or CH2-0-R' wherein R' represents a phenyl, hydroxyphenyl or tolyl group or a straight chain alkyl group having from 1 to 6 carbon atoms. The process of the invention, using enzymatic hydrolysis, allows the final product to be obtained, un- der conditions remarkably plainer than those used in the chemical hydrolysis, in very high yields and with a drastic reduction of the quantity of undesired by-products. Hydrolasic enzymes suitable for the present process are, for example, acid or neutral proteasis which under appropriate conditions hydrolyze the amidic bond of an N-acyl-peptide with the formation of the desired peptide.

Particularly, preferred hydrolasic enzymes for the process of enzymatic hydrolysis of N-acyl-aspartame are deacylating enzymes obtained from microbic strains of Escherichia, Nocardia, Proteus, Penicillium, 50 Aspergillus or Streptomyces genus, commonly classified as acylasis and more specifically as Penicillin acylasis.

The hydrolytic process can be carried out either by using directly the free or immobilized microbic cells or by isolating the specific enzymes which c9n be used in the free form, immobilized according to known techniques to resins, glass, cellulose or similar substances by ionic or covalent bonds or grafted to fibres 55 permeable to the substrate, or insolubilized by cross-linkage. Immobilization or insolubilization is advan tageous as the same enzyme can be used for many production cycles. The use of the enzymes isolated and purified to the desired degree is preferred rather than the raw cellular extract since the extraction or purification process normally allows a reduction or elimination of the presence of contaminated enzymes which could lower the yields by formation of undesired by-products.

Some examples of suitable enzymes are those of fungal origin acylasis producted by fermentation of various species of actinomycetes, filamentous fungi and yeasts, such as 2 GB 2 160 870 A 2 Aiternatia Mucor Aspergillus Penicillium Botrytis Phoma Cephalosporium Streptomyces Cryptococcus Trichoderma Emericellopsis Trichophyton Epicoccum Trichosporon Epidermophyton Actinoplanes Fasarium Nocardia 10 or those of bacterial origin acylasis produced by fermentation of various bacteric species such as Aerobacter Flavobacterium Alcaligenes Micrococcus Bordetella Proteus 15 Cellulomonas Pseudomonas Corynebacterium Salmonella Erwinia Sarcina Escherichia Zanthomonas B-megaterium B.subtilis 20 Achromobacter Also enzymatic preparations obtained by extraction of animal organs, such as pig-kidney acylasis are able to cause hydrolysis of the amidic bond between the carbonyl group of an organic acid and the aminic group of Laspartyi-L-phenylaianine methyl ester.

The enzymes may be added to an aqueous. solution of from 0.2 to 260 g/l of the N-acyl derivative, suitably mildly buffered at different pH according to the enzyme used, that is in a range of from 2 to 9, preferably from 5 to 7. The reaction may be carried out at a temperature of from 10' to 600C, preferably 15' to 400C, for 1 to 48 hours operating in batch or column according to the quantity of the enzyme present in the reaction mixture and to the ratio between the quantity of the enzyme in solution or in the 30 immobilized form and the quantity of N-substituted dipeptide present in the reaction mixture. The yields of the reactions carried out under optimal conditions reach values higher than 80%. The extraction of the product may be carried out using known techniques, but complicated and expensive processes for recovering the unreacted compounds or the undesired by-products may be avoided.

The following Examples and Preparation illustrate the invention.

Other N-acyl derivatives, the preparation of which is not specifically indicated, were obtained in high yields by operating as described in the preparation A, B, C and D and using a suitable acylating derivative as starting material.

Preparation A N-Phenylacetyl-L-up-aspartyl 1 phenylalanine methyl ester 66.6 g of L-aspartic acid were suspended in 100 ml of water. 200 mi of 10% sodium hydroxide were added and the mixture was cooled to W- 17'C. A mixture of 100 mi of 10% sodium hydroxide and 70 mi of phenylacetyl chloride was dropped therein for 1 hour. The mixture was kept for 3 hours at 2WC and then 37% hydrochloric acid was added until the pH was 2. After cooling to 0' -5'C and filtration, the reac- 45 tion mixture was dried to obtain N-phenylacetyl-aspartic acid (120 g), 99. 7% titre.

78 g thereof were suspended in 80 m] of acetic anhydride. The mixture was heated to 800 for 2 hours.

g of N-phenylacetyl-aspartic anhydride were obtained after cooling and precipitation with a solvent.

52 g of N-phenylacetyl-aspartic anhydride were suspended in 52 mi of glacial acetic acid and 100 m[ of dichloroethane, and the suspension was cooled to 100C. 200 m] of dichloroethane containing 40.2 9 of 50 phenylalanine methyl ester was added. 85 g of the title compound we. re obtained after extractions with basis adjusting the pH to acid value and filtration.

PreparationB 55 N-Phenoxyacetyl-L-(xasparty.'-L-phenylalanine methyl ester Operating as described in Preparation A, but using 77.2 g of phenoxyacetyl chloride instead of the phenylacetyl chloride, 110 g ol the title compound were obtained.

Preparation C 60 N-p-hydroxyphenylaceL.yl-L-otp-aspartyl-L-phenylalanine methyl ester Operating as described in Preparation A, but using 60.4 9 of p- hydroxyphenylacetyl chloride instead of the phenylacetyl chloride, 70.7 g of the title compound were obtained.

GB 2 160 870 A 3 Preparation D N-propionyl-L-u.p-aspartyl-L-phenylalanine methyl ester Operating as described in Preparation A, but using 41.8 g of propionyl chloride instead of the phenylacetyl chloride, 33.8 g of the title compound were obtained. The acylasis enzyme compounds were pre5 pared by fermentation with abovementioned microorganisms in specific culture broths.

Preparation E Acylasis enzyme from E. Coli A TCC 11105 The microorganism was fermented in a culture broth consisting of yeast extract, sodium glutammate, phenyl-acetic acid and ammonium salts, buffered so as to have an initial pH of 6.8-6.9. The optimal growth was reached in 10-12 hours with cellulasis dried weight of 300-400 mg/1 00 mi of broth. When the fermentation was over, the cells were separated by centrifugation, washed and passed on to a lysis treatment with n-butyl acetate. The lysate compound was flocculated, clarified and ultrafiltered. The concentrate obtained by selective ultrafiltration was salted with ammonium sulphate. The salted compound consisted of a stable acylasis having an activity of 600-900 Ulmi.

EXAMPLE 1

2 g of N-phenylacetyi-L-aspartyi-L-phenylaianine methyl ester (obtained as described in Preparation A) was dissolved in 100 mi of water with progressive additions of 10% sodium hydroxide, keeping the mix ture under shaking at 370C and at a pH lower than 7.5. When the solution was over, 1 N sodium hydrox- 20 ide or 1 N hydrochloric acid was added to stabilize the pH at 6.5-6 and the mixture was added with 0.5 g of the enzyme compound having an acylasic activity of 50 Ulg, obtained by immobilization of the com pound of Preparation E on a solid substrate, by selective adsorption and cross-linkage with glutaralde hyde, in the presence of albumin.

The mixture was allowed to react for 23 hours at 37'C under shaking, the pH being kept at 6.t0.5 by 25 addition of 1 N sodium hydroxide. At the end of the reaction the immobilized enzyme was separated off by filtration in vacuo through a glass filter and washed with 100 mi of water. The filtrate and the washing liquors were combined and the solution was analysed by high pressure liquid chromatography (HPLC) indicating a hydrolysis yield of N-phenylacetyl- L-a-aspartyi-L- phenylaianine methyl ester of 90% of the theoretic value only 5% of the non-reacted compound and 5% of undesired products. The obtained as- 30 partame was then extracted and crystallized according to suitable preparation processes from low salt containing aqueous solutions, and the free phenylacetic acid was recovered by extraction with solvents or adsorption on resins and used again for further preparations.

EXAMPLE2

2g of N-phenoxyacetyl-L-aspartyl-L-phenylalanine methyl ester (obtained as described in Preparation B) was dissolved in 100 ml of water at 35'C with addition of 10% sodium hydroxide to keep the pH not higher than 7.5. After dissolution the pH was adjusted to 6.0 and 0.5 g of Novozym acylasis 217 at 60 U/g were added, the enzyme being immobilized on a suitable insoluble substrate. The mixture was allowed to react for 24 hours at 35'C under shaking maintaining the pH at 6:LO.5 with addition of 1 N sodium hydroxide, The immobilized enzyme was then recovered by filtration through a glass filter and washed with 10 ml of water. The filtrate and the washing liquors were combined and the solution was chromatographed (HPLC) obtaining a yield of 93% of the theoretic value.

EXAMPLE 3

The reaction was carried out as described in Example 1, except that the enzyme was adsorbed on an ion exchange resin and insolubilised by crosslinkage in the presence of gluteraidehyde and a diamine according to known methods [Hayes R., Biochem. Biophys. Res. Commun 36, 235 (1969)]. The compound having 16 U/g was used in a quantity of 2 g per 100 ml of 2% solution of N-phenylacetyl-L-aspartyl-L- phenylalanine methyl ester. Reaction yields were 80% after 22 hours of reaction.

EXAMPLE 4

The reaction was carried out as described in Example 1, except that the enzyme was insolubilized by grafting onto cellulose acetate fibres according to known processes [Dinelli D., Process Biochem 718,9, (1972)1. The compound had 10.000 Ulg and 1 g of fibre was used to obtain 90% transformation under the 55 conditions of Example 1.

EXAMPLE 4 Bis

The reaction was carried out as described in Example 1, except that the enzyme was insolubilized on porous glass particles with primary aminic functions by activation with gluteraldehyde and a covalent 60 bond of the enzyme on the carbonylic intermediate. The compound had 25 U/g and 2 g of the immobi lized enzyme was used to obtain 75% transformation under the conditions of Example 1.

4 GB 2 160 870 A EXAMPLE 5

The reaction was carried out as described in Example 1, except that the enzyme of preparation E was used in the soluble form. 160 U of enzyme extracted from culture broths of E. Coli was added to 100 m[ of a 2% solution of N-phenylacetyi-L-asparty]-L-phenylalanine methyl ester. The reaction mixture was maintained for 8 hours at 37'C and pH 6-6.5. The yield of the desired product was 87% of the theoretic value.

4 EXAMPLE 6

3 g of N-p-hldroxyphenylacetyl-L-aspartyl-L-phenylalanine methyl ester (obtained as described in Prep- aration Q were dissolved in 100 ml of water, the pH was adjusted to 6 and the solution was heated to 10 40'C. 1 g of acylasis (penicillinacylasis) from E. Coli adsorbed on an ion exchange resin and having the activity of 40 UIg was then added. The reaction mixture was kept under shaking, at 400C, a pH 5.5-6.5 for 20 hours. The immobilized enzyme was then separated off by filtration. The reaction mixture analyzed by the HPLC showed an 82% conversion of the starting material to aspartame.

EXAMPLE 7

Example 6 was repeated except that the enzyme was obtained by extraction of the culture broth of Nocardia and was used in the soluble form. By carrying out the reaction under the described conditions, N-phydroxyphenylacecyl-L-aspartyl-L-phenylaianine methyl ester was transformed into aspartame with a yield of 50% of the theoretic value.

EXAMPLE 8

Example 1 was repeated except that the immobilized enzyme was obtained by extraction from a culture broth oi Proteus, had an activity of 25 Ulg, and was used in a quantity of 1 91100 mi of the reaction mixture. The aspartame yield obtained under these conditions was 48% of the theoretic value.

is EXAMPLE 9

3 g of N-propionyl-L-aspartyl-L-phenylalanine methyl ester obtained according to Preparation D were dissolved in 100 ml of water. The solution was heated to 37'C and the pH was adjusted to 6.5. To the reaction mixture was added 300 U/g of the enzymatic compound obtained by extraction and purification 30 according to known methods of the culture broth of E. Coli. The reaction mixture was kept under shaking for 24 hours at the indicated temperature, keeping the pH at 6.5 0.5. The final yield of aspartame was higher than 40% of the theoretic value.

EXAMPLE 70

Operating as described in Example 9, but substituting a 0.5 g/l solution of N-undecylearbonyl-L-aspartyl-L-phenylalanine methyl ester for the Npropionyl derivative and keeping the pH at 6 1, gave a 30% yield of aspartame.

EXAMPLE 11

2 g of N-formyi-L-aspartyl-L-phenylaianine methyl ester were dissolved in 100 mI of water at 370C. When solution was complete, the pH was adjusted to 6.5-7 with 10% sodium hydroxide and 250 U of an enzymatic compound having acylasic activity obtained from Pseudomonas culture was added. The reaction mixture vjas allowed to stand for 36 hours at 370C. The pH was then adjusted to 4-4.5 and the corn- pound was separated by concentration. The yield of L-ct-aspartyl-L- phenylalanine methyl ester was 20% 45 of the theoretic value.

EXAMPLE 12

Operating as in Example 2, except that an enzyme extracted from broth culture of Aspergillus and hav % ing 18.000 W9 was used at a concentration of 2% of that of N-phenoxy- methyi-L-aspartame, with hydrol- 50 ysis yield of 20% of the theoretic value.

EXAMPLE 13

As described in Example 1, except for the enzyme was not isolated, but the cells are immobilized "in toto": 1 g of humid cells obtgined from E. Coli culture was suspended in 5 mi of buffer phosphate 50 5E mM, pH 7.5. The solution was added with 0.5% of bovine albumin and 1% of glutaraldehyde. After 2 hours at room temperature the obtained pellets were separated by filtration and washed with buffer phosphate 50 mM. The compound used had an activity of 14 Ulg and was used in a quantity of 2 g per mi of a 20,6 solution of 1,1 -phenylacety]-L-aspa rtyl- L-phenylalanine methyl ester. The transformation yields after 8 hours of incubation at 37'C were 65% of the theoretic value.

EXAMPLE 141

As described in Example 2, except for the enzyme was not isolated and the mycelium mass obtained from a Streptomyces culture was immobilized. The linkage process was analogous to that described in Example 13. The cross-iinked mycelium mass was separated by filtration, washed with water and lyophi- 61 GB 2 160 870 A 5 lized. The Iyophilized compound was roughly triturated and used for the selective hydrolysis of N-phenoxyacetyi-L-aspartyi-L-phenylaianine methyl ester. 2 g of the compound having an activity of 12 U/g was used per 100 mi of 2% solution of the product to be hydrolysed. The transformation yields after 8 hours of incubation at 37'C are 60% of the theoretic value.

EXAMPLE 15

1 g of N-propo)cyacetyl-L-aspartyl-L-phenylaianine methyl ester was dissolved in 100 ml of water at 37'C, adjusting the pH to 6 with 10% sodium hydroxide. To the reaction mixture was added 250 U of the enzymatic compound having acylasic activity obtained from Cephalosporium culture. The mixture was allowed to stand for 20 hours at 37oC. At the end of the enzymatic reaction the yield was 45% of the theoretic value.

EXAMPLE 16 1 g of N-tolylo)cyacetyl-L-aspartyl-L-phenylalanine methyl ester was dissolved in 100 ml of water. 10% sodium hydroxide was added progressively to pH 6-7. To the mixture was added 250 U of enzymatic compound having acylasic activity obtained from Actinoplanes Utahensis cultures. The reaction was carried out at 37'C for 24 hours. The hydrolysis yield of the N-tolyloxyacetyl derivative was 40% of the theoretic value.

Claims

1. A process for the preparation of an ester of L-ct-aspartyi- Lphenylalanine having the general formula 1 COOR, 1 H,N-CH-CO-NH-CH-CH2-C,H, 1 C1-12-COOH (1) wherein R, represents an alkyl group having from 1 to 4 carbon atoms, the process comprising enzymatically hydrolyzing an N-acyl derivative of the desired product, which N-acyl derivative has the general formula 11 COOP, i R-CO-NI-I-CH-CO-NH-CH-CH2-C^ 1 H2COOH wherein R represents a free or substituted alkyl, alkenyl or phenylalkyl group having from 1 to 11 carbon atoms and R, is as above defined.

2. A process according to claim 1 in which R, represents a methyl group.

3. A process according to claim 1 or claim 2 in which R represents a hydrogen atom or a group of the formula -CH3, -(CH.),CH, wherein n is a number of from 1 to 10, -C,H,, - CH2-C,H,, - CH2-C,H,-OH, -CH2-C,H.N021 -C,H,(OCH3)2, -CH2-C,H,(OH)2, -CH2-CH=CH-CH,-CH,-CH,-S-CH,-CH=CH2 or -CH2-0-R' wherein R' repre sents a phenyl, hydroxyphenyl or tolyl group or a straight chain alkyl group having from 1 to 6 carbon atoms.

4. A process according to any preceding claim in which the enzymatic hydrolysis is carried out using a hydrolasic enzyme.

5. A process according to claim 4 in which the hydrolasic enzyme is prepared from a microorganism.

6. A process according to claim 5 in which the microorganism is an actinomycete, fungus or bacterium.

7. A process according to claim 6 in whi.;h the actinomycete is of the Nocardia, Actinoplanes or Streptomyces genus.

8. A process according to claim 6 in which the fungus is of the Alternatia, Aspergillus, Botrytis, Cephalosporium, Cryptococcus, Emericellopsis, Epicoccum, Epidermophyton, Fusarium, Mucor, Penicillium, Phoma, Tritoderma, T richophyton or Trichosphoron genus.

9. A process according to claim 6 in which the bacterium is of the Aerobacter, Alcaligenes, Bordetella, Cellulomonas, Corynebacterium, Erwinia, Escherichia Achromobacter, Flavobacterium, Micrococcus, Proteus, Pseudomonas, Salmonella, Sarcina, Eanthomonas or B. subtilis genus.

10. A process according to any preceding claim in which the enzymatic hydrolysis is carried out either in the presence of microbic cells producing the desired enzyme or in the presence of a separately prepared enzyme.

6 GB 2 160 870 A 6

11. A process according to claim 10 in which the enzymatic hydrolysis is carried out in the presence of an enzyme prepared separately, isolated and purified.

12. A process according to claim 10 or claim 11 in which the enzyme or the microbic cells producing it are either immobilized on an inert substrate or insolubilized by a cross-linkage process.

13. A process according to any preceding claim in which the enzymatic hydrolysis is carried out in aqueous solution at a concentration of from 0.2 to 260 g/l, buffered at a pH of from 2 to 9, at a temperature of from 10' to 60T.

14. A process according to claim 13 in which the aqueous solution is buffered at a pH of from 5 to 7.

15. A process according to claim 13 or claim 14 in which the enzymatic hydrolysis is carried out at a 10 temperature of from 15 to 40T.

Printed in the UK for HMSO, D8818935, l 1185, 7102. Published by The Patent Office, 25 Southampton Buildings, London, WC2A lAY, from which copies may be obtained.